Monoclonal antibody which specifically binds to but does not activate the CD8+ cells

ABSTRACT

This invention provides a method of isolating CD8 +  cells which employs an antibody which specifically binds to CD8 molecules present on the surface of CD8 +  cells but does not activate the CD8 +  cells once bound. This invention also provides related hybridoma cell lines, monoclonal antibodies, antigenic polypeptides, isolated CD8 +  cells, and kits.

This application is a divisional application of U.S. patent applicationSer. No. 09/521,527, filed on Mar. 8, 2000, now U.S. Pat. No. 6,790,662,which claims priority from U.S. provisional application No. 60/124,253,filed on Mar. 12, 1999.

Throughout this application, various publications are cited. Thedisclosure of these publications is hereby incorporated by referenceinto this application to describe more fully the state of the art towhich this invention pertains.

FIELD OF THE INVENTION

This invention relates to a positive selection method for isolating CD8⁺cells using certain CD8-specific antibodies. The isolated CD8⁺ cellshave importance as vehicles for combating viral infections and tumors.

BACKGROUND OF THE INVENTION

In humans, CD8⁺ cells play a vital role in the immune system's abilityto defend against potentially harmful foreign entities, such as bacteriaand viruses [1]. CD8⁺ cells circulate in the blood and possess on theirsurface the CD8 protein. When necessary, these cells are converted intocytotoxic cells (i.e. cell-killing cells) which proceed to destroyforeign cells, viruses, and other harmful pathogens present in thesubject [2]. Because of CD8⁺ cells' effective role in host defense, theyhold great potential in isolated form as therapeutics for treatingdisorders such as viral infections and malignancies [3].

In the past, purification of human CD8+ cells has been achieved bynegative selection. Specifically, peripheral blood mononuclear cells(“PBMC's”) are incubated with a cocktail of monoclonal antibodiesspecific for non-CD8 sub-populations. These sub-populations include, forexample, B-cells, CD4⁺ cells, NK cells, macrophages and neutrophils, andeach contains specific, non-CD8 “markers”. The sub-populations are thenremoved from the resulting antibody cocktail using magnetic beads [4].This technique has certain major disadvantages. The first is thatseveral monoclonal antibodies are required for removing non-CD8⁺ cells.The second is that the resulting CD8⁺ population suffers fromcontamination from non-CD8⁺ cells that possess relatively low levels ofnon-CD8 markers. Finally, when a magnetic separation procedure is usedto remove all non-CD8⁺ cells, a large number of magnetic beads areneeded.

SUMMARY OF THE INVENTION

This invention provides a method of isolating CD8⁺ cells which comprisedthe steps of

-   -   (a) contacting a sample of isolated peripheral mononuclear blood        cells with a first antibody which specifically binds to the        sequence AAEGLDTQRFSG (SEQ ID NO: 1), or portion thereof, on CD8        molecules present on the surface of CD8⁺ cells but does not        activate the CD8⁺ cells once bound thereto, under conditions        permitting the formation of a first complex between the CD8⁺        cell and first antibody;    -   (b) separating from the sample any first antibody not present in        the resulting first complex;    -   (c) contacting the sample with a second, immobilized antibody        which specifically binds to the first antibody in the first        complex, under conditions permitting the formation of an        immobilized, second complex between the first complex and the        second antibody, thereby immobilizing the CD8⁺ cells present in        the sample;    -   (d) separating from the resulting immobilized second complex the        cells present in the sample which were not immobilized in step        (c);    -   (e) contacting the immobilized second complex under suitable        conditions with an agent which causes the dissociation of the        second complex into CD8⁺ cells and an immobilized third complex        between the first antibody and second antibody; and    -   (f) separating the immobilized third complex from the CD8⁺        cells, thereby isolating the CD8⁺ cells.

This invention also provides a hybridoma cell line which produces amonoclonal antibody which specifically binds to CD8 molecules present onthe surface of CD8⁺ cells but does not activate the CD8⁺ cells. Thisinvention further provides monoclonal antibodies produced by each of theinstant hybridoma cell lines. Finally, this invention provides relatedpolypeptides, isolated CD8⁺ cells and kits.

DETAILED DESCRIPTION OF THE INVENTION

The hybridoma cell lines designated 37B1 and 8G6 were deposited on Dec.11, 1997 and Mar. 4 1999, respectively, pursuant to, and in satisfactionof, the requirements of the Budapest Treaty on the Internationalrecognition of the Deposit of Microorganisms for the Purposes of PatentProcedure with the American Type Culture collection (ATCC), 10801University Boulevard, Manassas, Va. 2010-2209 under ATCC Accession Nos.HB-12441 and HB-12657, respectively.

This invention provides a, method of isolating CD8⁺ cells by employingan anti-CD8 antibody, along with certain other reagents. Specifically,this invention provides a method of isolating CD8⁺ cells which comprisesthe steps of

-   -   (a) contacting a sample of isolated peripheral mononuclear blood        cells with a first antibody which specifically binds to the        sequence AAEGLDTQRFSG (SEQ ID NO: 1), or portion thereof, on CD8        molecules present on the surface of CD8⁺ cells but does not        activate the CD8⁺ cells once bound thereto, under conditions        permitting the formation of a first complex between the CD8⁺        cell and first antibody;    -   (b) separating from the sample any first antibody not present in        the resulting first complex;    -   (c) contacting the sample with a second, immobilized antibody        which specifically binds to the first antibody in the first        complex, under conditions permitting the formation of an        immobilized, second complex between the first complex and the        second antibody, thereby immobilizing the CD8⁺ cells present in        the sample;    -   (d) separating from the resulting immobilized second complex the        cells present in the sample which were not immobilized in step        (c);    -   (e) contacting the immobilized second complex under suitable        conditions with an agent which causes the dissociation of the        second complex into CD8⁺ cells and an immobilized third complex        between the first antibody and second antibody; and    -   (f) separating the immobilized third complex from the CD8⁺        cells, thereby isolating the CD8⁺ cells.

As used herein, a “CD8⁺ cell” means a T-cell having on its surface theCD8 protein. In the preferred embodiment, the CD8⁺ cells are human CD8⁺cells. The CD8⁺ cells can be from any CD8⁺ cell-possessing species.“Isolating” CD8⁺ cells means obtaining a population of peripheralmononuclear blood cells wherein the ratio of CD8⁺ cells to non-CD8⁺cells is at least about 7:1. In the preferred embodiment of thisinvention, this ratio is at least about 9:1.

This invention employs several types of antibodies which specificallybind to given epitopes. More particularly, this invention uses a “firstantibody” which specifically binds to the sequence AAEGLDTQRFSG (SEQ IDNO: 1), or portion thereof, on CD8 molecules present on the surface ofCD8⁺ cells but does not activate the CD8⁺ cells once bound thereto.Here, CD8⁺ cell “activation” means causing CD8⁺ cells to expressγ-interferon (“γ-IFN”). This activation can be measured using routinemethods such as sandwich ELISA assays, which can be performed usingcommercially available kits.

Such first antibodies include, but are not limited to, the monoclonalantibodies produced by the hybridoma cell lines 37B1 (ATCC Accession No.HB-12441) and 8G6 (ATCC Accession No. HB-12657). Conditions which permitthese antibodies to bind to but not activate CD8⁺ cells are well knownin the art. These conditions include for example, in a suitable buffersuch as . . . Ca2⁺ and Mg2⁺-free Dulbecco's Phosphate Buffer Saline(DPBS) containing 1% Human Serum Albumin (HSA) and 0.2% sodium citrateand gentle mixing by “end over end” rotation on a rotator set at 4 rpm.

As used herein, the term “antibody” includes, but is not limited to,both naturally occurring and non-naturally occurring antibodies.Specifically, the term “antibody” includes polyclonal and monoclonalantibodies, and binding fragments thereof. Furthermore, the term“antibody” includes chimeric antibodies and wholly synthetic antibodies,and fragments thereof. In one embodiment, the antibody is a monoclonalantibody. The monoclonal antibody can be human, or that of anotherspecies including, for example, mouse and rabbit. In this invention, anantibody which “specifically” binds to a stated epitope binds to thatepitope with a dissociation constant of at least about 10-fold less thanthe dissociation constant with which it binds to any other epitope. Inone embodiment, this dissociation constant ratio is at least about 100.In the preferred embodiment, this dissociation constant ratio is atleast about 10³.

The “second antibody” used in the instant method can be any antibodywhich specifically recognizes an epitope on any portion of the firstantibody. In the preferred embodiment, the second antibody specificallyrecognizes a portion of the constant (Fc) region of the first antibody.Such anti-Fc antibodies are commercially available and include, forexample, sheep anti-mouse antibody immobilized on magnetic beads [5].

The agent that causes dissociation of the immobilized second complexinto CD8⁺ cells and immobilized antibodies can be any agent whichsuccessfully competes with the CD8 molecule for specific binding to thefirst antibody. In the preferred embodiment, this agent is thepolypeptide designated CD8-3 having the sequence AAEGLDTQRFSG (SEQ IDNO: 1). In one embodiment, the immobilized second antibody comprises anantibody operably affixed to a magnetic bead.

This invention also provides a hybridoma cell line which produces amonoclonal antibody which specifically binds to CD8 molecules present onthe surface of CD8⁺ cells but does not activate the CD8⁺ cells. In oneembodiment, the hybridoma cell line is selected from the cell linesdesignated 37B1 (ATCC Accession No. HB-12441) and 8G6 (ATCC AccessionNo. HB-12657). This invention further provides the monoclonal antibodiesproduced by each of the instant hybridoma cell lines.

This invention further provides a polypeptide useful for generating theinstant monoclonal antibody that comprises the amino acid sequenceAAEGLDTQRFSG (SEQ ID NO: 1). In the preferred embodiment, thepolypeptide is the polypeptide designated CD8-3 and having the aminoacid sequence AAEGLDTQRFS (SEQ ID NO: 2). The instant polypeptide canoptionally comprise one or more additional amino acid residues at theC-terminal or N-terminal end. In the preferred embodiment, thepolypeptide has the sequence NKPKAAEGLDTQRFSGKRLG (SEQ ID NO: 3).

This invention further provides a population of CD8⁺ cells isolated bythe instant method.

Finally, this invention provides a kit for use in isolating CD8⁺ cellswhich comprises, in separate compartments, (a) an antibody whichspecifically binds to the sequence AAEGLDTQRFSG (SEQ ID NO: 1), orportion thereof, on CD8 molecules present on the surface of CD8⁺ cells,but does not activate the CD8⁺ cells once bound thereto; and (b) anagent which causes the dissociation of a CD8⁺ cell-antibody complex. Inone embodiment, the agent which causes the dissociation of a CD8⁺cell-antibody complex comprises the polypeptide having the sequenceAAEGLDTQRFSG (SEQ ID NO: 1). In the preferred embodiment, the agent isthe polypeptide consisting of the sequence AAEGLDTQRFSG (SEQ ID NO: 1).

The instant kit can further comprise reagents useful for performing thebinding and dissociation steps of the instant method. The components ofthe instant kit can either be obtained commercially or made according towell known methods in the art. In addition, the components of theinstant kit can be in solution or lyophilized as appropriate. In thepreferred embodiment, the kit further comprises instructions for use.

The following procedures relating to the instant invention are routinein the art: isolating peripheral mononuclear blood cells from which theCD8⁺ cells are in turn isolated [6]; separating unbound antibodies andcells from a sample containing bound antibodies and/or cells viacentrifugation or spinning membrane; and immobilizing antibodies viapolystyrene flasks, columns or beads [4,7].

This invention will be better understood by reference to theExperimental Details which follow, but those skilled in the art willreadily appreciate that the specific experiments detailed are onlyillustrative of the invention as described more fully in the claimswhich follow thereafter.

Experimental Details

Rationale

Human CD8⁺ cells can be isolated from preparations of peripheral bloodmononuclear cells (PBMC's) by either positive or negative selection.Positive selection results in a highly-purified population of CD8⁺cells. Negative selection, while resulting in sufficient numbers of CD8⁺cells, has low levels of contaminating non-CD8 populations remainingafter the selection procedure.

The idea was to generate an antibody which has high affinity for CD8⁺cells, does not activate the cells during the selection process, and iscapable of being easily eluted from the cells. An anti-peptide antibodyappeared to meet these criteria. However, it was known that anti-peptideantibodies might be of low affinity and may recognize the linear peptidesequence exclusively, preventing reactivity with native antigen.

It was necessary that the anti-CD8 antibody not activate the cellsduring the selection process, as it would lessen their ability toeffectively act as naïve responder cells during in vitro stimulationprotocols. The use of peptide release to selectively isolate a cellpopulation has been show by Tseng-Law, et al. [8] for CD34⁺ cells.

Methods

The CD8 alpha chain was examined for hydrophilic sequences and fourpeptides selected. All were coupled to keyhole limpet hemocyanin (KLH)as carrier and used to immunize mice. A C-terminal amino acid was addedto each of the peptides coupled to KLH to make the monoclonalantibodies. Antisera from the mice were evaluated for the ability torecognize both peptide and native CD8 on the surface of T cells. Onlyone of the four peptides was capable of recognition of both antigenicforms of CD8. Monoclonal antibodies were generated to this peptide andthe resulting antibody used to isolate CD8⁺ cells from a PBMCpreparation. The antibody was successful in isolating a population ofhighly-purified CD8⁺ cells (Table 1) which were not activated by theisolation procedure (Table 2).

TABLE 1 Purification of CD8⁺ Cells by Positive Selection Analyzed byFlow Cytometry* PBMC POST SELECTION % Fluorescence % Fluorescence CELLTYPE (Range) (Range) CD8 T cells 15 (7-24)  82 (56-95) CD4 T cells 36(14-52)   2 (0.1-10) CD14 Monocytes 15 (7-26) 0.8 (0.2-2) CD15Neutrophils 12 (8-21) 0.6 (0.1-3) CD19 B cells  2 (0.4-7)   3 (0.5-9)CD56 NK cells  6 (2-17)   6 (0.1-20) *Summary of 10 normal donors

TABLE 2 Activation of CD8⁺ Cells Isolated By Negative or PositiveSelection (Assessed by IFNγ Production) Negative Selection PositiveSelection Cells (pg/ml) (pg/ml) un-stimulated 20 20 allo-stimulation1440 3600

REFERENCES

-   1. Nabholz M. and H. R. MacDonald (1983) Annual Review of Immunology    1:273-306.-   2. Riddell S. R. and P. D. Greenberg (1994) Current Topics in    Microbiology and Immunology 189:9-34.-   3. Riddell S. R. and P. D. Greenberg (1995) Annual Review of    Immunology 13:545-586.-   4. Horgan K and S. Shaw (1994) Current Protocols in Immunology    2:7.4.1.-   5. Lea T, et al. (1988) Journal of Molecular Recognition 1(1):9-18.-   6. Kanof, M. E., et al. (1994) Current Protocols in Immunology    2:7.1.1.-   7. Kanof M. E. (1994) Current Protocols in Immunology 2:7:3:1.-   8. PCT International Publication No. WO 95/34817.

1. A hybridoma cell line selected from the group consisting of the cellline designated 37B1 (ATCC Accession No. HB-1244) and the cell linedesignated 8G6 (ATCC Accession No. HB-12657).
 2. The monoclonal antibodyproduced by the hybridoma cell line of claim 1.